Presenting stretched strands of single strand DNA for sequencing double strand DNA

ABSTRACT

A mechanism is provided for presenting single strands of a double strand molecule to a membrane. The double strand molecule is driven to a first side of the membrane by an electric field. The membrane has a first and second nanopore spaced apart by a nanopore separation distance. The first strand of the double strand molecule is captured in the first nanopore when driven to the first side of the membrane. The second strand is captured in the second nanopore by having the nanopore separation distance between the first nanopore and the second nanopore corresponding to a strand separation distance between the first and second strands, and/or by having captured the first strand to limit diffusion of the second strand. The first and second strands respectively in the first and second nanopores are individually stretched, by the first and second strands recombining on the second side of the membrane.

DOMESTIC PRIORITY

This application claims priority to U.S. Provisional Application Ser.No. 61/986,364, filed Apr. 30, 2014, which is incorporated herein byreference in its entirety.

BACKGROUND

The present invention relates to nanopore devices, and morespecifically, to presenting single strands of DNA or RNA molecules to amembrane from double strands of DNA or RNA.

Nanopore sequencing is a method for determining the order in whichnucleotides occur on a strand of deoxyribonucleic acid (DNA). A nanopore(also referred to a pore, nanochannel, hole, etc.) can be a small holein the order of several nanometers in internal diameter. The theorybehind nanopore sequencing is about what occurs when the nanopore issubmerged in a conducting fluid and an electric potential (voltage) isapplied across the nanopore. Under these conditions, a slight electriccurrent due to conduction of ions through the nanopore can be measured,and the amount of current is very sensitive to the size and shape of thenanopore. If single bases or strands of DNA pass (or part of the DNAmolecule passes) through the nanopore, this can create a change in themagnitude of the current through the nanopore. Other electrical oroptical sensors can also be positioned around the nanopore so that DNAbases can be differentiated while the DNA passes through the nanopore.

The DNA can be driven through the nanopore by using various methods, sothat the DNA might eventually pass through the nanopore. The scale ofthe nanopore can have the effect that the DNA may be forced through thehole as a long string, one base at a time, like thread through the eyeof a needle. Recently, there has been growing interest in applyingnanopores as sensors for rapid analysis of biomolecules such asdeoxyribonucleic acid (DNA), ribonucleic acid (RNA), protein, etc.Special emphasis has been given to applications of nanopores for DNAsequencing, as this technology holds the promise to reduce the cost ofsequencing below $1000/human genome.

SUMMARY

According to an embodiment, a method is provided for presenting singlestrands of a double strand molecule to a membrane. A double strandmolecule is driven to a first side of the membrane by an electric field,where the membrane includes a first nanopore and a second nanoporespaced apart by a nanopore separation distance. The membrane includes asecond side opposite the first side. The double strand moleculecomprises a first strand and a second strand when driven to the firstside of the membrane. The first strand of the double strand molecule iscaused to be captured in the first nanopore by the electric field whenthe double strand molecule is driven to the first side of the membrane.The second strand of the double strand molecule is caused to be capturedin the second nanopore by having the nanopore separation distancebetween the first nanopore and the second nanopore corresponding to astrand separation distance between the first strand and the secondstrand, by having captured the first strand to limit diffusion of thesecond strand, and/or both by having the nanopore separation distancebetween the first nanopore and the second nanopore corresponding to astrand separation distance between the first strand and the secondstrand and by having captured the first strand to limit diffusion of thesecond strand. The first strand in the first nanopore and the secondstrand in the second nanopore are individually stretched, which iscaused by the first strand and the second strand recombining on thesecond side of the membrane.

According to an embodiment, a system is provided for presenting singlestrands of a double strand molecule to a membrane. The system includes amembrane comprising a first side opposite a second side opposite, wherethe double strand molecule driven to the first side of the membrane byan electric field from a voltage source. The membrane includes a firstnanopore and a second nanopore spaced apart by a nanopore separationdistance. A first reservoir is on the first side. The double strandmolecule is introduced on the first side and comprises a first strandand a second strand when driven to the first side of the membrane. Thevoltage source causes the first strand of the double strand molecule tobe captured in the first nanopore by the electric field when the doublestrand molecule is driven to the first side of the membrane. The secondstrand of the double strand molecule is captured in the second nanoporeby having the nanopore separation distance between the first nanoporeand the second nanopore corresponding to a strand separation distancebetween the first strand and the second strand, by having captured thefirst strand to limit diffusion of the second strand, and/or both byhaving the nanopore separation distance between the first nanopore andthe second nanopore corresponding to a strand separation distancebetween the first strand and the second strand and by having capturedthe first strand to limit diffusion of the second strand. A secondreservoir is on the second side, where the first strand in the firstnanopore and the second strand in the second nanopore are individuallystretched by the first strand and the second strand recombining on thesecond side of the membrane in the second reservoir.

Additional features and advantages are realized through the techniquesof the present invention. Other embodiments and aspects of the inventionare described in detail herein and are considered a part of the claimedinvention. For a better understanding of the invention with theadvantages and the features, refer to the description and to thedrawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The subject matter which is regarded as the invention is particularlypointed out and distinctly claimed in the claims at the conclusion ofthe specification. The forgoing and other features, and advantages ofthe invention are apparent from the following detailed description takenin conjunction with the accompanying drawings in which:

FIG. 1 is a schematic of a dual nanopore/nanochannel presentation anddetection system according to an embodiment.

FIG. 2 is a schematic of the system which shows an example of additionalelements that may be utilized to individually sequence one single strandand individually sequence another single strand translocating throughrespective nanopores according to an embodiment.

FIG. 3 illustrates a bow tie DNA molecule ligated to the double strandmolecule in which formation of a first fork and second fork of a bow tieend promote individual capture in the nanopores according to anembodiment.

FIG. 4 is a flowchart of a method for respectively presenting two singlestrands of a double strand molecule to a membrane for individualsequencing according to an embodiment.

FIG. 5 is a block diagram that illustrates an example of a computer(computer test setup) having capabilities, which may be included inand/or combined with embodiments.

DETAILED DESCRIPTION

Sequencing of DNA with nanopore technologies requires the presentationof single strand DNA to the base sensing module. One particularchallenge is to present the single strand in stretched configuration tothe base sensing module. This stretching is required to provide maximumspatial separation of DNA bases of the single strand. Failure to achievestretching allows multiple bases to simultaneously interact with thebase sensing module, thereby reducing the fidelity and resolution ofsequencing.

An embodiment provides a technique and mechanism for presentingstretched strands of single strand DNA for the purpose of sequencingdouble strand (stranded) DNA.

Now turning to the figures, FIG. 1 is a schematic of a system 100according to an embodiment. The system 100 includes a nanodevice 150.The nanodevice 150 includes a membrane 101. The membrane 101 includes ananopore 102 and a nanopore 103. In one case, the nanopores 102 and 103may each be nanochannels, and it is contemplated that nanochannels andany nano-opening can be utilized as discussed herein according toembodiments as would be understood to one skilled in the art. Eachnanopore 102 and 103 may be 1 to 2 nanometers (nm) in nanopore diameteror nanochannel size (e.g., width).

The membrane 101 may be silicon, silicon dioxide, or any insulatingmaterial. Also, the nanopores 102 and 103 may be the combination of twoattached proteins.

A nanopore separation distance 104 is shown as the distance separatingthe nanopore 102 from the nanopore 103 in the membrane 101. A topreservoir 160 (cis or source side) is sealed to the top side of themembrane 101 and a bottom reservoir 165 (trans or sink side) is sealedto the bottom side of the membrane 101. Electrode 111 is in the topreservoir 160 and electrode 112 is in the bottom reservoir 165.Electrode 111 is connected to a negative side of a voltage source 115and electrode 112 is connected to the positive side of the voltagesource 115. An electrically conductive solution 170 fills the topreservoir 160, bottom reservoir 165, nanopore 102, and nanopore 103.

As an example, a double strand DNA molecule 107 may be introduced intothe top reservoir 160 by typical means such as a syringe, pump, etc. Thedouble strand DNA molecule 107 may be referred to as the target moleculefor testing.

Double stranded DNA is normal and abundant, e.g., genomic DNA is almostentirely double stranded. An embodiment of the present invention allowsdirect handling and sequencing of double stranded DNA molecules, andeliminates the sample preparation steps (e.g., fragmentation andmelting) required for single strand sequencing devices. The embodimentallows for sequencing of virtually unlimited lengths of DNA, sincedouble strand DNA is stable for virtually unlimited lengths, which is anadvantage over single strand sequencing devices where the read lengthsare limited by stability of single strand DNA molecules.

Operation of the nanodevice 150 in the system 100 translocates doublestrand DNA from the top reservoir 160 (source) to the bottom reservoir165 (sink). Unwinding (i.e., base pair opening on source side) andhelical rewinding (i.e., base pairing on sink side) is discreet (i.e.,base pair by base pair); therefore, the nanodevice 150 achievesratcheting of double stranded DNA through the device.

The double strand DNA molecule 107 has a single strand DNA molecule 105and a single strand DNA 106. The single strand DNA molecule 105 has asequence that is complementary to the sequence of single strand DNA 106.This causes the two strands to hybridize into double stranded DNA 108 incertain normal conditions (e.g., temperature below melting temperature).

Hybridization of single strands into double strand configuration on bothsides (source side and sink side) of the membrane 101 naturally extendsalong the length of the available molecules, to a physical proximity ofpore openings of nanopores 102 and 103. As the double strand DNAmolecule 107 in the top reservoir 160 is driven to the membrane 101, thesingle strand DNA 105 is captured in nanopore 102 (for example) and theother single strand DNA 106 is capture in the other close by nanopore103. As the energetics of hybridization favor double strandconfiguration over single strand configuration, the hybridizationextends to as close a proximity of nanopores 102 and 103 because of thesmall nanopore separation distance 104 and the flexibility of stretchedsingle strand region 109 of single strand DNA 105 and stretched singlestrand region 110 of single strand DNA 106. The energetics naturallydrive for a minimization of stretched single stranded regions 109 and110, thereby stretching the single strand DNA 105 at the stretchedsingle strand region 109 and stretching the single strand DNA 106 at thestretched single strand region 110 respectively through the nanopores102 and 103.

Further details regarding operation of the system 100 are now discussed.The double strand DNA molecule 107 is introduced in the top reservoir160. The double strand DNA molecule 107 is negatively charged. Thedouble strand DNA molecule 107 is presented on the source side of themembrane 101. For example, the voltage source 115 applies a positivevoltage to electrode 112 and negative voltage to electrode 111, whichgenerates an ionic current through the electrically conductive solution170, and an upward pointing electric field (resulting in a downwardforce on the double strand DNA molecule 107). The negative voltage anddownward force translocate the negatively charged double strand DNAmolecule 107 from the source side of the membrane to the sink sidethrough the openings which may be nanopores/nanochannels 102 and 103 inthe membrane 101. The electrical field induced by the voltage source 115(i.e., the circuit of the electrodes 111 and 112, the voltage source115, and electrically conductive solution 170) causes capture of onestrand such as the single strand DNA 105 in one nanopore/channel such asthe nanopore 102.

The capture of the single strand DNA 105 in nanopore 102 limits thediffusion of the other single strand DNA 106 to the proximity of thenanopore 102, as the molecular region of the source side is hybridizedas the double strand DNA molecule 107. This limitation of diffusion ofthe single strand DNA 106 provides an enhanced capture rate for thesingle strand DNA 106 to be captured by the electric field throughnanopore 103 (because the single strand DNA 106 is confined to the closeproximity of nanopore 102 which is only separated from nanopore 103 bynanopore separation distance 104 which can be a few nanometers). Thesingle strand DNA 105 is bound to the single strand DNA 106 at the pointof hybridization (on the double strand DNA molecule 107), and thisprevents the single strand DNA 106 from leaving the area of the nanopore103.

Accordingly, the single strand DNA 106 is captured in the nanopore 103while the single strand DNA 105 is in the nanopore 102 (e.g., the singlestrand DNA 105 is being threaded through the nanopore 103 as the voltageof the voltage source 115 is applied). Now, as the electrical field fromthe voltage of the voltage source 115 connected to electrodes 111 and112 translocates the two single strands 105 and 106 through respectivenanopores 102 and 103, these strands (single strand 105 and singlestrand 106) move into the sink side of membrane 101 in bottom reservoir165.

As the length of the two molecules (i.e., single strand DNA 105 andsingle strand DNA 106) on the sink side exceeds the minimum required(e.g., usually 10-12 bases on each strand) the two strands (singlestrand DNA 105 and single strand DNA 106) hybridize on the sink side inthe bottom reservoir 165. The hybridized single strand DNA 105 andsingle strand DNA 106 is labeled as 108, which is now a double strandDNA molecule 108 on the sink side in the bottom reservoir 165.

Accordingly, the single strand DNA molecules 105 and 106 are the doublestrand DNA molecule 107 on the source side. The single strand DNAmolecules 105 and 106 individually translocate through nanopores 102 and103 respectively, and then hybridize (rewind) to form the double strandDNA molecule 108. As noted above, the energetics of hybridization favordouble strand configuration over single strand configuration. Thehybridization (rewinding) on the sink side continuously forming thedouble strand DNA molecule 108 (until the double strand DNA molecule 107on the source side has been fed through the nanopores 102 and 103)causes the stretched single strand region 109 of single strand DNA 105and the stretched single strand region 110 of single strand DNA 106.This stretching (of the single strand DNA molecules 105 and 106) occursbecause of the hybridization (rewinding) pulls against the force of theunwinding by the double strand DNA molecule 107. Pulling in oppositedirections occurs while the single strand DNA molecule 105 is beingstretched in nanopore 102 and the single strand DNA molecule 106 isbeing stretched in nanopore 103. Since the energetics naturally drivefor a minimization of single stranded region 109 and 110, the singlestands 105 and 106 are straightened out in these regions for basesequencing (as discussed in an example in FIG. 2.

The following options to improve initial capture of the single strandDNA molecule 105 in the nanopore 102 (in this example). The doublestrand DNA molecule 107 terminates with a single strand overhang (i.e.,a few nanometers). For example, the single strand DNA 105 is longer thansingle strand DNA 106 of the double strand DNA molecule 107. Thisavailability of unpaired single strand DNA enhances the initial captureof single strand 105 into nanopore 102 or 103.

As another option to increase capture of the single strand DNA molecule105 and/or the single strand DNA molecule 106, the double strand DNAmolecule 107 terminates with non-complementary ends (i.e., withnon-complementary bases). This is achieved by enzymatic ligation ofshort oligomeric non-complementary DNA fragments to single strand 105and single strand 106. For example, (A)_10 attached to the terminals ofboth strands will create a frayed non-hybridized terminal that willalways remain single strand and increase the efficiency of capture ofeach single strand DNA molecule (ssDNA) 105 and 106 into the respectivenanopores 102 and/or 103 (respectively). In other words, the terminatingends of the single strand 105 and single strand 106 may end with thesame (non-complementary) base.

As another option, FIG. 3 shows a bow tie DNA molecule 300 according toan embodiment. The double strand DNA molecule 107 terminates with Yshaped double stranded ends (i.e., forked ends). This is achieved byligation of custom synthesized Y shaped DNA fragments to single strands105 and 106. This increases the physical separation distance “D” betweenterminal ends (i.e., between single strands 105 and 106), and allows thenanodevice 150 to function with larger a nanopore separation distance104.

Another advantage is that nanopore 102 and 103 can also be larger, andthese need to be size selective to double stranded DNA. For example, thenanopores 102 and 103 can have an approximately (˜) 10 nm diameter whenthe bow tie DNA molecule 300 is utilized. When the bow tie DNA molecule300 is not ligated to the double strand molecule 107, the nanopores 102and 103 may be less than 2 nm (e.g., 1 nm).

The bow tie DNA 300 has a bow tie end on each end. The bow tie end hastwo forks, shown as fork A and fork B (which respectively correspond tosingle strands 105 and 106 in FIGS. 1 and 2). Operation of the system200 applies for the forks A and B as discussed herein for single strandDNA 105 and 106 respectively.

The forks A (single strand DNA 105) and B (single strand DNA 106) have aseparation distance D that is matched (e.g., equal to or nearly equal tothe nanopore separation distance 104). The forks A and B are connectedto a stem (double strand DNA). The stem of the bow tie end is ligated tothe double strand DNA molecule 107 for sampling. Note that the optionsto increase capture rate discussed above also apply for the bow tie DNA300. For example, forks A and B may terminate with non-complementaryends (i.e., with non-complementary bases) and/or fork A may have anoverhang as compared to fork B, both of which enhance the capture of thesingle strands 105 and 106.

Further information regarding the bow tie DNA is found in filed patentapplication “Bow Tie DNA Compositions And Methods” IBM®, Application No.61/986,343 (concurrently filed with Application No. 61/986,364), whichis herein incorporated by reference in its entirety.

According to an embodiment, FIG. 2 is a schematic of the system 100which shows an example of additional elements that may be used toindividually sequence the single strand DNA 105 and sequence the singlestrand DNA 106 translocating through the nanopores 102 and 103. Someelements in FIG. 1 are omitted from the system 200 in FIG. 2 so as notto obstruct the view, but it is understood that the omitted elements arepresent in the system 200 as discussed herein.

In FIG. 2, a pair of electrodes 215 a and 215 b are in the nanopore 102and a pair of electrodes 220 a and 220 b are in the nanopore 103. Thepair of electrodes 215 a and 215 b are connected to a voltage source 210a and ammeter 205 a. The pair of electrodes 220 a and 220 b areconnected to a voltage source 210 b and ammeter 205 b.

As the stretched single strand DNA 105 passes through the nanopore 102,a base of the single strand 105 is sequenced by applying voltage of thevoltage source 210 a and by measuring the tunneling current (via ammeter205 a) interacting with the base (in the nanopore 102). Each individualbase of the single strand DNA 105 (in the stretched region 109) passingthrough the nanopore 102 can be identified by its respective tunnelingcurrent (i.e., change in tunneling current according to the particularbase in the nanopore) as understood by one skilled in the art. Likewise,the complementary base on the single strand DNA 106 in the nanopore 103is identified (sequenced) by applying voltage of the voltage source 210b and then measuring (via ammeter 205 b) the tunneling current.Accordingly, each individual base of the single strand DNA 106 (in thestretched single strand region 110) passing through the nanopore 103 canbe identified by its respective tunneling current.

By having the single strand DNA 105 and single strand DNA 106 stretchedin respective stretched single strand regions 109 and 110 allows asingle base to be in respective nanopores 102 and 103 (each one) at atime, when the sample is the double strand molecule 107. This allows thedouble strand molecule 107 to be presented as single strands forsequencing without having to change the double strand molecule 107 intotwo separate (non-connected) strands.

FIG. 4 is a flowchart of a method 400 for presenting single strands of adouble strand molecule 107 (e.g., target molecule) to a membrane 101according to an embodiment. Reference can be made to FIGS. 1-3 discussedherein (along with FIG. 5 below).

Voltage of the voltage source 115 drives the (negatively charged) doublestrand molecule 107 to the first side of the membrane 101 by an electricfield at block 405. The membrane 101 includes a first nanopore 102 and asecond nanopore 103 spaced apart by the nanopore separation distance104. The second side (sink side) of the membrane 101 is opposite thefirst side (source side).

The double strand molecule 107 includes a first single strand 105 and asecond single strand 106 when driven to the first side of the membrane101 in the top reservoir 160 at block 410.

Voltage of the voltage source 115 causes the first single strand 105 ofthe double strand molecule 107 to be captured in the first nanopore 102by the electric field (e.g., pointing upward) when the double strandmolecule 107 is driven to the first side of the membrane 101 at block415.

While the voltage of the voltage source 115 is applied and while thefirst single strand 105 of the double strand molecule 107 is beingthread into the nanopore 102, the second single strand 106 of the doublestrand molecule 107 is captured in the second nanopore 103 by having thenanopore separation distance 104 between the first nanopore 102 and thesecond nanopore 103 corresponding to a strand separation distance (D)between the first single strand 105 and the second single strand 106, byhaving captured the first single strand 105 to limit diffusion of thesecond single strand 106, and/or by both at block 420.

The first single strand 105 in the first nanopore 102 and the secondsingle strand 106 in the second nanopore 103 are individually stretchedby (the force/pull of) the first single strand 105 and the second singlestrand 106 recombining (into the double strand molecule 108) on thesecond side of the membrane 101 in the bottom reservoir 165 at block425.

The first strand in the first nanopore and the second strand in thesecond nanopore are single strands of the double strand molecule 107that are stretched.

The first single strand 105 and the second single strand 106 recombiningon the second side (bottom reservoir 165) of the membrane 101 includesthe first single strand 105 and the second single strand 106 undergoinghelical rewinding on the second side into a second double strandmolecule 108 in the bottom reservoir 165. The energetics ofhybridization drive the first single strand 105 and the second singlestrand 106 to undergo helical rewinding on the second side of themembrane 101. The hybridization of the first single strand 105 and thesecond single strand 106 on the second side generates a pull (e.g.,downward force) on the first single strand 105 while in the firstnanopore 102 and on the second single strand 106 while in the secondnanopore 103. The pull by the hybridization on the second side isopposite a force (upward force) of unwinding of the first single strand105 and the second single strand 106 of the double strand molecule 107to cause the first single strand 105 and the second single strand to bestretched while in the first nanopore 102 and the second nanopore 103respectively.

The nanopore separation distance 104 and the strand separation distance(D in FIG. 3) are substantially equal, and/or the nanopore separationdistance 104 is smaller than the strand separation distance (D).

The first single strand 105 has an overhang (i.e., is longer) comparedto the second single strand 106, and the overhang of the first strandenhances an initial capture of the first strand in the first nanopore102.

A bow tie end (bow tie DNA 300) is ligated to the double strand molecule107. The bow tie end comprises a first fork, a second fork, and a doublestrand stem ligated to the double strand molecule 107 (as shown in FIG.3). The first single strand 105 is the first fork and the second singlestrand 106 is the second fork of the bow tie end ligated to the doublestrand molecule 107.

The first single strand 105 has a terminating end that isnon-complementary to a terminating end of the second single strand 106.

The double strand molecule 107 is a deoxyribonucleic acid and/or is aribonucleic acid. The bases of the first single strand 105 in the firstnanopore 102 and bases of the second single strand 106 in the secondnanopore 103 are sequenced, e.g., via respective tunneling currentsmeasured by ammeters 205 a and 205 b. Bases of the first single strand105 in the first nanopore 102 are sequenced concurrently while bases ofthe second single strand 106 in the second nanopore 103 are sequenced.

Note that a nanopore (also referred to a pore, nanochannel, hole, etc.)can be a small hole in the order of several nanometers (e.g., 4-8 nm) ininternal diameter and/or the nanopore can be a larger hole in the orderof a few microns (μm) in internal diameter. The theory behind nanoporesequencing is about what occurs when the nanopore is submerged in aconducting fluid (such as conductive solution 170) and an electricpotential (voltage) is applied across the nanopore (e.g., via voltagesource 115). The length of the nanopore corresponds to the thickness ofthe membrane 101 traversed by the nanopore 102. In one case,nanochannels are simply considered as nanopores of larger length. Thesize of the membrane 101 and the channels/nanopores 102 and 103 can beeither small or quite large. When the membrane and correspondingchannels/nanopores are large, the thickness of the membrane 101 andlength of the nanopores 102, 103 may each be, e.g., 1-5 microns (μm).When the membrane and corresponding channels/nanopores are small, thethickness of the membrane 101 and length of the nanopores 102, 103 mayeach be, e.g., 4-8 nm.

FIG. 5 illustrates an example of a computer 500 (e.g., as part of thecomputer test setup for testing and analysis) which may individuallyimplement, control, and/or regulate the voltage of the voltage sources115, 210 a, 210 b, and measurements of the ammeters 205 a and 205 b asdiscussed herein.

Various methods, procedures, modules, flow diagrams, tools,applications, circuits, elements, and techniques discussed herein mayalso incorporate and/or utilize the capabilities of the computer 500.Moreover, capabilities of the computer 500 may be utilized to implementfeatures of exemplary embodiments discussed herein. One or more of thecapabilities of the computer 500 may be utilized to implement, toconnect to, and/or to support any element discussed herein (asunderstood by one skilled in the art) in FIGS. 1-4. For example, thecomputer 500 which may be any type of computing device and/or testequipment (including ammeters, voltage sources, connectors, etc.).Input/output device 570 (having proper software and hardware) ofcomputer 500 may include and/or be coupled to the nanodevices andstructures discussed herein via cables, plugs, wires, electrodes, patchclamps, etc. Also, the communication interface of the input/outputdevices 570 comprises hardware and software for communicating with,operatively connecting to, reading, and/or controlling voltage sources,ammeters, and current traces (e.g., magnitude and time duration ofcurrent), etc., as discussed herein. The user interfaces of theinput/output device 570 may include, e.g., a track ball, mouse, pointingdevice, keyboard, touch screen, etc., for interacting with the computer500, such as inputting information, making selections, independentlycontrolling different voltages sources, and/or displaying, viewing andrecording current traces for each base, molecule, biomolecules, etc.

Generally, in terms of hardware architecture, the computer 500 mayinclude one or more processors 510, computer readable storage memory520, and one or more input and/or output (I/O) devices 570 that arecommunicatively coupled via a local interface (not shown). The localinterface can be, for example but not limited to, one or more buses orother wired or wireless connections, as is known in the art. The localinterface may have additional elements, such as controllers, buffers(caches), drivers, repeaters, and receivers, to enable communications.Further, the local interface may include address, control, and/or dataconnections to enable appropriate communications among theaforementioned components.

The processor 510 is a hardware device for executing software that canbe stored in the memory 520. The processor 510 can be virtually anycustom made or commercially available processor, a central processingunit (CPU), a data signal processor (DSP), or an auxiliary processoramong several processors associated with the computer 500, and theprocessor 510 may be a semiconductor based microprocessor (in the formof a microchip) or a macroprocessor.

The computer readable memory 520 can include any one or combination ofvolatile memory elements (e.g., random access memory (RAM), such asdynamic random access memory (DRAM), static random access memory (SRAM),etc.) and nonvolatile memory elements (e.g., ROM, erasable programmableread only memory (EPROM), electronically erasable programmable read onlymemory (EEPROM), programmable read only memory (PROM), tape, compactdisc read only memory (CD-ROM), disk, diskette, cartridge, cassette orthe like, etc.). Moreover, the memory 520 may incorporate electronic,magnetic, optical, and/or other types of storage media. Note that thememory 520 can have a distributed architecture, where various componentsare situated remote from one another, but can be accessed by theprocessor 510.

The software in the computer readable memory 520 may include one or moreseparate programs, each of which comprises an ordered listing ofexecutable instructions for implementing logical functions. The softwarein the memory 520 includes a suitable operating system (O/S) 550,compiler 540, source code 530, and one or more applications 560 of theexemplary embodiments. As illustrated, the application 560 comprisesnumerous functional components for implementing the features, processes,methods, functions, and operations of the exemplary embodiments.

The operating system 550 may control the execution of other computerprograms, and provides scheduling, input-output control, file and datamanagement, memory management, and communication control and relatedservices.

The application 560 may be a source program, executable program (objectcode), script, or any other entity comprising a set of instructions tobe performed. When a source program, then the program is usuallytranslated via a compiler (such as the compiler 540), assembler,interpreter, or the like, which may or may not be included within thememory 520, so as to operate properly in connection with the O/S 550.Furthermore, the application 560 can be written as (a) an objectoriented programming language, which has classes of data and methods, or(b) a procedure programming language, which has routines, subroutines,and/or functions.

The I/O devices 570 may include input devices (or peripherals) such as,for example but not limited to, a mouse, keyboard, scanner, microphone,camera, etc. Furthermore, the I/O devices 570 may also include outputdevices (or peripherals), for example but not limited to, a printer,display, etc. Finally, the I/O devices 570 may further include devicesthat communicate both inputs and outputs, for instance but not limitedto, a NIC or modulator/demodulator (for accessing remote devices, otherfiles, devices, systems, or a network), a radio frequency (RF) or othertransceiver, a telephonic interface, a bridge, a router, etc. The I/Odevices 570 also include components for communicating over variousnetworks, such as the Internet or an intranet. The I/O devices 570 maybe connected to and/or communicate with the processor 510 utilizingBluetooth connections and cables (via, e.g., Universal Serial Bus (USB)ports, serial ports, parallel ports, FireWire, HDMI (High-DefinitionMultimedia Interface), etc.).

In exemplary embodiments, where the application 560 is implemented inhardware, the application 560 can be implemented with any one or acombination of the following technologies, which are each well known inthe art: a discrete logic circuit(s) having logic gates for implementinglogic functions upon data signals, an application specific integratedcircuit (ASIC) having appropriate combinational logic gates, aprogrammable gate array(s) (PGA), a field programmable gate array(FPGA), etc.

As used herein, polynucleotides include DNA and RNA, and are polymeric,contiguous, i.e., covalently bonded, strands of nucleotides.

“Complementary” or “substantially complementary” refers to thehybridization or base pairing or the formation of a duplex betweennucleotides or nucleic acids, such as, for instance, between the twostrands of a double-stranded DNA molecule or between an oligonucleotideprimer and a primer binding site on a single stranded nucleic acid.Complementary nucleotides are, generally, A and T (or A and U), or C andG. Two single stranded RNA or DNA molecules are said to be substantiallycomplementary when the nucleotides of one strand, optimally aligned andcompared and with appropriate nucleotide insertions or deletions, pairwith at least about 80% of the nucleotides of the other strand, usuallyat least about 90% to 95%, and more specifically about 98 to 100%.Alternatively, substantial complementarity exists when an RNA or DNAstrand will hybridize under selective hybridization conditions to itscomplement. Typically, selective hybridization will occur when there isat least 65% complementary over a stretch of at least 14 to 25nucleotides, specifically at least about 75%, more specifically at leastabout 90% complementary.

The term “single stranded DNA” (ssDNA) as used herein refers to anaturally occurring or synthetic deoxyribonucleic acid moleculecomprising a linear single strand, for example, a ssDNA can be a senseor antisense gene sequence.

“Duplex” is used interchangeably with “double-stranded” and means atleast two oligonucleotides and/or polynucleotides that are fully orpartially complementary and that undergo Watson-Crick type base pairingamong all or most of their nucleotides so that a stable complex isformed. The terms “annealing” and “hybridization” are usedinterchangeably to mean the formation of a stable duplex. In one aspect,stable duplex means that a duplex structure is not destroyed by astringent wash, e.g., conditions including temperature of about 5° C.less that the T_(m) (melting temperature) of a strand of the duplex andlow monovalent salt concentration, e.g., less than 0.2 M, or less than0.1 M. “Perfectly matched” in reference to a duplex means that the poly-or oligonucleotide strands making up the duplex from a doubleWatson-Crick basepairing with a nucleotide in the other strand. The term“duplex” includes the pairing of nucleoside analogs, such asdeoxyinosine, nucleosides with 2-aminopurine bases, PNAs, and the like,that may be employed. A “mismatch” in a duplex between twooligonucleotides or polynucleotides means that at pair of nucleotides inthe duplex fails to undergo Watson-Crick bonding.

“Hybridization” refers to the process in which two single-strandedpolynucleotides bind non-covalently to form a stable double-stranded orduplex polynucleotide. The term “hybridization” may also refer totriple-stranded hybridization. The resulting (usually) double-strandedpolynucleotide is a “hybrid” or “duplex.” “Hybridization conditions”will typically include salt concentrations of less than about 1 M, moreusually less than about 500 mM or less than about 200 mM. Hybridizationtemperatures can be as low as 5° C., but are typically greater than 22°C., more typically greater than about 30° C., and specifically in excessof about 37° C.

Hybridizations are usually performed under stringent conditions, i.e.,conditions under which a probe will specifically hybridize to its targetsubsequence. Stringent conditions are sequence-dependent and aredifferent in different circumstances. Longer fragments may requirehigher hybridization temperatures for specific hybridization. As otherfactors may affect the stringency of hybridization, including basecomposition and length of the complementary strands, presence of organicsolvents and extent of base mismatching, the combination of parametersis more important than the absolute measure of any one alone. Generally,stringent conditions are selected to be about 5° C. lower than the T_(m)for the specific sequence at a defined ionic strength and pH. Exemplarystringent conditions include salt concentration of at least 0.01 M to nomore than 1 M Na ion concentration (or other salts) at a pH 7.0 to 8.3and a temperature of at least 25° C. For example, conditions of 5×SSPE(750 mM NaCl, 5.0 mM sodium phosphate, 5 mM EDTA, pH 7.4) and atemperature of 25-30° C. are suitable for allele-specific probehybridizations. “Hybridizing specifically to” or “specificallyhybridizing to” or like expressions refer to the binding, duplexing, orhybridizing of a molecule substantially to or only to a particularnucleotide sequence or sequences under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA.

The term “blunt end” as used herein refers to the end of a dsDNAmolecule having 5′ and 3′ ends, wherein the 5′ and 3′ ends terminate atthe same nucleotide position. Thus, the blunt end comprises no 5′ or 3′overhang.

“Ligation” means to form a covalent bond or linkage between the terminiof two or more nucleic acids, e.g. oligonucleotide and/orpolynucleotide. Ligation included blunt-end ligation as well as ligationwith a single strand overhang. The nature of the bond or linkage mayvary widely and the ligation may be carried out enzymatically orchemically. In one embodiment, ligations are carried out enzymaticallyto form a phosphodiester linkage between a 5′ carbon of a terminalnucleotide of one oligonucleotide with 3′ carbon of anotheroligonucleotide. Examples of ligases include Taq DNA ligase, T4 DNAligase, T7 DNA ligase, and E. coli DNA ligase. The choice of the ligasedepends to a certain degree on the design of the ends to be joinedtogether. Thus, if the ends are blunt, T4 DNA ligase may be employed,while a Taq DNA ligase may be preferred for a sticky end ligation, i.e.a ligation in which an overhang on each end is a complement to eachother.

As used herein, a “target polynucleotide” or “target DNA” is apolynucleotide from a sample. In one embodiment, a target polynucleotideis a double stranded polynucleotide (e.g., DNA) for which the nucleotidesequence is to be determined.

A sample may be collected from an organism, mineral or geological site(e.g., soil, rock, mineral deposit, combat theater), forensic site(e.g., crime scene, contraband or suspected contraband), or apaleontological or archeological site (e.g., fossil, or bone) forexample. A sample may be a “biological sample,” which refers to amaterial obtained from a living source or formerly-living source, forexample, an animal such as a human or other mammal, a plant, abacterium, a fungus, a protist or a virus. The biological sample can bein any form, including without limitation a solid material such as atissue, cells, a cell pellet, a cell extract, or a biopsy, or abiological fluid such as urine, blood including plasma or serum, saliva,amniotic fluid, exudate from a region of infection or inflammation, or amouth wash containing buccal cells, urine, cerebral spinal fluid andsynovial fluid and organs.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of onemore other features, integers, steps, operations, element components,and/or groups thereof.

The corresponding structures, materials, acts, and equivalents of allmeans or step plus function elements in the claims below are intended toinclude any structure, material, or act for performing the function incombination with other claimed elements as specifically claimed. Thedescription of the present invention has been presented for purposes ofillustration and description, but is not intended to be exhaustive orlimited to the invention in the form disclosed. Many modifications andvariations will be apparent to those of ordinary skill in the artwithout departing from the scope and spirit of the invention. Theembodiment was chosen and described in order to best explain theprinciples of the invention and the practical application, and to enableothers of ordinary skill in the art to understand the invention forvarious embodiments with various modifications as are suited to theparticular use contemplated

The flow diagrams depicted herein are just one example. There may bemany variations to this diagram or the steps (or operations) describedtherein without departing from the spirit of the invention. Forinstance, the steps may be performed in a differing order or steps maybe added, deleted or modified. All of these variations are considered apart of the claimed invention.

While the preferred embodiment to the invention had been described, itwill be understood that those skilled in the art, both now and in thefuture, may make various improvements and enhancements which fall withinthe scope of the claims which follow. These claims should be construedto maintain the proper protection for the invention first described.

What is claimed is:
 1. A system for presenting single strands of adouble strand molecule to a membrane, the system comprising: a membranecomprising a first side opposite a second side opposite, the doublestrand molecule driven to the first side of the membrane by an electricfield from a voltage source, the membrane comprising a first nanoporeand a second nanopore spaced apart by a nanopore separation distance; afirst reservoir on the first side, the double strand molecule introducedon the first side and comprising a first strand and a second strand whendriven to the first side of the membrane; wherein the voltage sourcecauses the first strand of the double strand molecule to be captured inthe first nanopore by the electric field when the double strand moleculeis driven to the first side of the membrane; wherein the second strandof the double strand molecule is captured in the second nanopore byhaving the nanopore separation distance between the first nanopore andthe second nanopore corresponding to a strand separation distancebetween the first strand and the second strand, by having captured thefirst strand to limit diffusion of the second strand, or both by havingthe nanopore separation distance between the first nanopore and thesecond nanopore corresponding to a strand separation distance betweenthe first strand and the second strand and by having captured the firststrand to limit diffusion of the second strand; and a second reservoiron the second side, the first strand in the first nanopore and thesecond strand in the second nanopore are individually stretched by thefirst strand and the second strand recombining on the second side of themembrane in the second reservoir.
 2. The system of claim 1, wherein thefirst strand in the first nanopore and the second strand in the secondnanopore are single strands and stretched.
 3. The system of claim 1,wherein the first strand and second strand recombining on the secondside of the membrane comprises the first strand and the second strandundergoing helical rewinding on the second side into a second doublestrand molecule in the second reservoir.
 4. The system of claim 3,wherein energetics of hybridization drive the first strand and thesecond strand to undergo the helical rewinding on the second side of themembrane.
 5. The system of claim 1, wherein the hybridization of thefirst strand and the second strand on the second side generates a pullon the first strand while in the first nanopore and on the second strandwhile in the second nanopore; wherein the pull by the hybridization onthe second side is opposite a force of unwinding of the first strand andthe second strand of the double strand molecule to cause the firststrand and the second strand to be stretched while in the first nanoporeand the second nanopore respectively.
 6. The system of claim 1, whereinthe nanopore separation distance and the strand separation distance aresubstantially equal.
 7. The system of claim 1, wherein the nanoporeseparation distance is smaller than the strand separation distance.